Project summary/abstract. The goal of this project is to define the role of the heme export protein Feline Leukemia Virus, subgroup C, Receptor (FLVCR) and heme signaling in hematopoiesis. Cats viremic with Feline leukemia virus, subgroup C (FeLV-C) down-regulate the cell surface expression of FLVCR and develop a profound macrocytic anemia. Mice in which Flvcr is deleted have a comparable clinical phenotype, and in both species, erythroid differentiation arrests at the CFU-E (colony-forming unit-erythroid)/proerythroblast stage. These data led us to hypothesize that FLVCR protects cells when heme synthesis precedes or exceeds globin synthesis and that if these steps were poorly coordinated, ineffective erythropoiesis and anemia would result. This grant will test our hypothesis by directly measuring free heme in developing erythroid cells with a luciferase reporter containing the MARE sequences (Bach1 binding site) of the -globin promoter and by measuring heme-regulated eIF2 kinase (HRI) and eIF2 phosphorylation. We will show that excess heme induces anemia with rescue experiments designed to restore the intracellular free heme balance of Flvcr-deleted mice by decreasing heme synthesis or by increasing heme degradation using methods independent of FLVCR. In studies of rps6+/- mice, we will determine whether this pathophysiology could be relevant to the macrocytic anemia of Diamond Blackfan anemia (results from RPS19 haploinsufficiency, failed ribosome assembly, and slowed translation initiation) and the 5q- myelodysplasia syndrome (acquired RPS14 deletion). We will also define the molecular pathways which trigger erythroid marrow failure by transcriptional profiling. In addition, we will explore the role of FLVCR in other hematopoietic cells using comparable experimental strategies, since our recent data suggest that FLVCR is needed for the maturation of double positive T cells to single positive T cells in thymus and may have a role in megakaryocyte endomitosis. We can thus determine whether these different lineages share regulatory pathways. Together these studies should provide significant insights into heme signaling and toxicities during hematopoietic cell differentiation.